E. faecalis V583 strain cells were grown at 37ºC in M17 0.5% glucose medium and collected at exponential phase (OD600 = 0.5) and at 24h stationary phase.
Extracted molecule
total RNA
Extraction protocol
For total RNA extraction cells were resuspended into 200 µl of “max bacterial enhancement reagent” (Invitrogen), transferred into micro tubes containing glass beads and 400 µl acid phenol (Ambion, Austin, TX) followed by mechanically lysation in Mixer Mill 200 (30/s, 30 min, Retsch, Haan, Germany). After centrifugation 10 min at 14,000 g at 4ºC, aqueous phase transferred to 2 ml tubes containing 1 ml Trizol reagent, 5 min incubation at room temperature (RT). Added 200 µl chloroform, mixed, incubated for 3 min at RT. Centrifugation for 15 min at 12,000 g at 4ºC; aqueous phase transferred into 2 ml tubes containing 200 µl chloroform, mixed gently and centrifuged again. RNAs contained in the Aqueous phase with RNAs precipitated by addition of 500 µl isopropanol, incubated for 10 min at RT. After centrifugation, RNA pellets washed with 75% ethanol, dried at RT. RNA pellets resuspended in DEPC-treated pure water. To enrich sncRNAs, 10µg RNA fractionated using flashPAGE Fractionator (Applied Biosystems, Foster City, CA).
Label
biotin
Label protocol
Fractionated RNA labelled using mirVana labelling kit (Applied Biosystems) hybridized onto the tiling array.
Hybridization protocol
Production, hybridization and data collecting were carried out by Febit biomed GmbH Company (Heidelberg, Germany).
Scan protocol
Production, hybridization and data collecting carried out by Febit biomed GmbH Company (Heidelberg, Germany) on the Febit Geniom platform.
Data processing
Genedata Expressionist software (version 5.3) was used for data processing. Raw data were Quantile normalized applying standard settings (Averaging: Logarithmic Mean) of Expressionist Analyst.
